Our OVERALL OBJECTIVE is to define the critical cellular mechanisms culminating in lethal liver cell injury. We will test the novel GENERAL HYPOTHESIS that NON-LYSOSOMAL PROTEOLYSIS IS AN ESSENTIAL MECHANISM MEDIATING LETHAL CELL INJURY DURING ANOXIA, OXIDATIVE STRESS AND BILE SALT CYTOTOXICITY. We will employ current and complementary biochemical and cell biological techniques to ascertain the regulatory and mechanistic aspects of proteolysis during lethal liver cell injury. The proposal has two SPECIFIC AIMS. FIRST, we will determine the cellular localization, regulation and identity of protease activity during lethal liver cell injury by directly testing: a) the HYPOTHESIS that proteolysis occurs during cell injury in all liver cell types, and occurs predominantly by nonlysosomal proteases, b) the HYPOTHESIS that proteolysis during cell injury is regulated by cellular ATP, cytosolic pH, vesicular pH and/or cytosolic free calcium; and c) the HYPOTHESIS that specific identifiable classes of proteases are responsible for the proteolysis occurring during cell injury. SECOND, we will determine the mechanistic role of proteolysis as a cause of lethal liver cell injury by directly testing: a) the HYPOTHESIS that proteolytic activity precedes the onset of cell death; and b) the HYPOTHESIS that inhibition of proteolytic activity delays the onset of lethal cell injury. The studies will employ homogeneous cultures and suspensions of isolated rat liver hepatocytes, sinusoidal endothelial cells and intrahepatic bile duct epithelial cells. In dispersed cell suspensions, total, lysosomal and non-lysosomal proteolysis, rates of cell killing, ATP depletion, and protease class specific proteolysis will be measured under basal conditions and during cell injury. Employing single cultured cells, fluorescent probes and multiparameter digitized video microscopy, cytosolic pH, vesicular pH, cytosolic free calcium, and protease class specific proteolysis will be quantitated using commercially available and novel synthetic fluorescent probes. Our LONG-TERM GOALS are to provide new fundamental information regarding proteolysis as a mechanism of lethal injury of liver cells during clinically relevant models of cell injury. Interventional strategies must be predicated on an understanding of the cellular mechanisms which lead to cell death. Successful completion of this proposal could identify a protease responsible for lethal liver cell injury. The results may lead to the identification of treatment modalities effective in the presentation of liver tissue during anoxia, oxidative stress, and cholestasis with retention of toxic bile salts.